Ramulus Cinnamomi extract attenuates neuroinflammatory responses via downregulating TLR4/MyD88 signaling pathway in BV2 cells

Ramulus Cinnamomi （RC）, a traditional Chinese herb, has been used to attenuate inflammatory responses. The purpose of this study was to investigate the effect of RC extract on lipopolysaccharide （LPS）-induced neuroinflammation in BV2 microglial cells and the underlying mechanisms involved. BV2 cells were incubated with normal medium （control group）, LPS, LPS plus 30 pg/mL RC extract, or LPS plus 100 pg/mL RC extract. The BV2 cell morphology was observed under an optical microscope and cell viability was detected by MTT assay. Nitric oxide level in BV2 cells was detected using Griess regents, and the levels of interleukin-6, interleukin-1 β, and tumor necrosis factor u in BV2 cells were determined by ELISA. The expression levels of cyclooxygenase-2, Toll-like receptor 4 and myeloid differentiation factor 88 proteins were detected by western blot assay. Compared with the LPS group, both 30 and 100 μg/mL RC extract had no significant effect on the viability of BV2 cells. The levels of nitric oxide, interleukin-6, interleukin-1β and tumor necrosis factor ct in BV2 cells were all significantly increased after LPS induction, and the levels were significantly reversed after treatment with 30 and 100 μg/mL RC extract. Furthermore, RC extract significantly inhibited the protein expression levels of cyclooxygenase-2, Toll-like receptor 4 and myeloid differentiation factor 88 in LPS-induced BV2 cells. Our findings suggest that RC extract alleviates neuroinflammation by downregulating the TLR4/MyD88 signaling pathway.

IL-1RⅠ/MyD88-TIR mimic AS-1 inhibits the activation of MyD88-dependent signaling pathway induced by IL-1β in vitro

Objective： To test whether IL-1 RI/My088-TIR mimic AS-1 can work as a new compound that targeted at blocking MyD88- dependent signaling pathway, we investigated the physical structure and biological function of AS-1. Methods：The crystallographic structure of AS-1 was examined by 1^H nuclear magnetic resonance. The toxicity of AS-1 was measured with Methyl thiazolyl tetrazolium （MTT） assay. The effect of AS-1 on phosphorylation state of p38 MAPK and IRAK-1 was observed with Western blot. Results：The crystallographic details of AS-1 demonstrated that it was a tri-peptide sequence[（F/Y）-（V/L/I）-（P/G）] of the IL-1R I -TIR domain BBloop. No toxicity of AS-1 was shown to HEK 293A cells. The phosphorylation of p38 MAPK, induced by IL-1β significantly increased from those in the control group. AS-1 significantly reduced the phosphorylation of p38 MAPK induced by IL-1β. IL-1β increased the phosphorylation of IRAK-1 significantly, which was prevented by AS-1. Conclusion：AS-1 is a competitive mimic between IL-1R I-TIR and MyD88-TIR domain, which most likely interferes with MyD88-dependent signaling pathway.

银翘柴桂Ⅱ号方对H1N1感染小鼠腹腔巨噬细胞MyD88-IRF7信号通路的影响

【目的】探讨银翘柴桂Ⅱ号方对H1N1感染小鼠腹腔巨噬细胞Ana-1中MyD88-IRF7信号通路的影响。【方法】应用银翘柴桂Ⅱ号方对H1N1感染小鼠腹腔巨噬细胞Ana-1干预24 h后,采用酶联免疫吸附法(ELISA)测定细胞上清液中白细胞介素6(IL-6)、肿瘤坏死因子α(TNF-α)水平,采用蛋白免疫印迹(Western blot)法检测骨髓样分化因子88(MyD88)、核因子kappa B(NF-κB)、干扰素调节因子7(IRF7)蛋白表达水平,采用反转录实时荧光定量聚合酶链反应(RT-qPCR)法检测白细胞介素1受体相关激酶4(IRAK4)m RNA表达水平。【结果】与H1N1感染组比较,银翘柴桂Ⅱ号方能显著下调Ana-1细胞IL-6、TNF-α水平(P<0.01),降低MyD88、NF-κB蛋白的表达水平(P<0.05或P<0.01),上调IRAK4 mRNA表达水平(P<0.01)。【结论】银翘柴桂Ⅱ号方可抑制H1N1所诱导的小鼠腹腔巨噬细胞My D88-IRF7信号通路以及NF-κB信号通路的活化,下调IL-6、TNF-α表达水平,发挥抗流感病毒的作用。

Correlation of MyD88 expression in the peripheral blood and placenta with the inflammatory response and insulin signal transduction in the placenta of patients with GDM

Objective: To study the correlation of MyD88 expression in peripheral blood and placenta with the inflammatory response and insulin signal transduction in the placenta of patients with gestational diabetes mellitus (GDM). Methods: The patients with GDM and healthy pregnant women who accepted antenatal care and gave birth in Guangyuan First People's Hospital between March 2015 and June 2017 were selected as the research subjects and enrolled in the GDM group and control group for the study respectively;the peripheral blood was collected before delivery to determine the MyD88 mRNA expression, and the placental tissue was collected after delivery to determine the mRNA expression of MyD88, inflammatory response molecules and insulin signal transduction molecules. Results: MyD88 mRNA expression levels in the peripheral blood and placenta of GDM group were significantly higher than those of control group, and the MyD88 mRNA expression in the peripheral blood was positively correlated with the MyD88 mRNA expression in the placenta;IL-1β, IL-6, RBP4, Chemerin, Resistin and PTP1B mRNA expression levels in the placenta of GDM group were significantly higher than those of control group whereas IRS1, ISR2, p-PI3K and GLUT4 protein expression levels were significantly lower than those of control group;IL-1β, IL-6, RBP4, Chemerin, Resistin and PTP1B mRNA expression levels in the placenta of GDM group of patients with high MyD88 expression were significantly higher than those of patients with low MyD88 expression whereas IRS1, ISR2, p-PI3K and GLUT4 protein expression levels were significantly lower than those of patients with low MyD88 expression. Conclusion:The expression of MyD88 in peripheral blood and placenta increase in patients with GDM and the change of MyD88 expression in peripheral blood could reflect the abnormality of inflammatory response and insulin signal transduction in the placenta.

ATF4 is directly recruited by TLR4 signaling and positively regulates TLR4-trigged cytokine production in human monocytes

Toll-like receptors （TLRs） are sentinels of the host defense system, which recognize a large number of microbial pathogens. The host defense system may be inefficient or inflammatory diseases may develop if microbial recognition by TLRs and subsequent TLR-triggered cytokine production are deregulated. Activating transcription factor 4 （ATF4）, a member of the ATF/CREB transcription factor family, is an important factor that participates in several pathophysiological processes. In this report, we found that ATF4 is also involved in the TLR-mediated innate immune response, which participates in TLR4 signal transduction and mediates the secretion of a variety of cytokines. We observed that ATF4 is activated and translocates to the nucleus following l ipopolysaccharide （LPS） stimulation via the TLR4-MyD88-dependent pathway. Additionally, a cytokine array assay showed that some key inflammatory cytokines, such as I L-6, I L-8 and RANTES, are positively regulated by ATF4. We also demonstrate that c-Jun directly binds to ATF4, thereby promoting the secretion of inflammatory cytokines. Taken together, these results indicate that ATF4 acts as a positive regulator in TLR4-triggered cytokine production.

Discovery of novel aporphine alkaloid derivative as potent TLR2 antagonist reversing macrophage polarization and neutrophil infiltration against acute inflammation

Toll-like receptor 2(TLR2)mediated macrophages regulate the protective immune response to infectious microorganisms,but the aberrant activation of macrophages often leads to pathological inflammation,including tissue damage.In this study,we identified antagonists of TLR2 by screening2100 natural products and subsequently identified Taspine,an aporphine alkaloid,as an excellent candidate.Furthermore,analysis of the 10 steps chemical synthesis route and structural optimization yielded the Taspine derivative SMU-Y6,which has higher activity,better solubility,and improved drug-feasible property.Mechanistic studies and seq-RNA analysis revealed that SMU-Y6 inhibited TLR2 over other TLRs,hindered the formation of TLR2/MyD88 complex,and blocked the downstream NF-κB and MAPK signaling pathway,thus suppressing the release of inflammatory cytokines.SMU-Y6 could stabilize TLR2 and bind to TLR2 protein with a Kdof 0.18μmol/L.Additionally,SMU-Y6 could efficiently reverse the M1 phenotype macrophage polarization,reduce the production of cytokines as well as infiltration of neutrophiles and alleviate the local inflammation in mice with acute paw edema and colitis.Collectively,we reported the first aporphine alkaloid derivative that selectively inhibits TLR2 with high binding affinity and superior drug-feasible property,thus providing an urgently-needed molecular probe and potential drug candidate for inflammatory and autoimmune disease therapy.